Reagents and cell culture

Chemical reagents, including DMSO, SDS, NaCl, and Tris base, were purchased from Sigma-Aldrich (St. Louis, MO). MG132 and cycloheximide (CHX) were obtained from Thermo Fisher Scientific (Waltham, MA). The DMEM, RPMI-1640 medium, and Fetal Bovine Serum (FBS) for cell culture were purchased from Invitrogen (Grand Island, NY). Human oral squamous cell carcinoma (OSCC) cells, including CAL27, SCC15, SCC25, and SCC9 were purchased from American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained according to ATCC protocols in a 37 °C humidified incubator with 5% CO₂. The ionizing radiation acquired resistance cell lines CAL27-IR and SCC25-IR were established in our laboratory by exposing CAL27 and SCC25 cells to gradually increasing dose of ionizing radiation for approximately 6 months. Briefly, CAL27 and SCC25 cells were serially irradiated with 2Gy of X-rays to a final dose of 80 Gy. Cells were cultured with DMEM medium and maintained at a 37 °C humidified incubator with 5% CO₂. The irradiated cells were passaged into new culture flask when growing to approximately 80% confluence. Reirradiation of Xrays was repeated over a period of 6 months (starting at 2 Gy and ending with 8 Gy for each irradiation, the dose was gradually increased for each month by 2 Gy), for a total dose of 80 Gy. Immortalized oral epithelial cell hTERT-OME was purchased from Applied Biological Materials (ABM) Inc. (Richmond, BC, Canada). The HaCat cells were purchased form Thermo Fisher Scientific (Waltham, MA). All cells were subjected to mycoplasma analysis every 2 months. Primary antibodies against Survivin (#2808), cleaved-PARP (#9532), VDAC1 (#4661), Bax (#14796), cleaved-caspase 3 (#9664), cytochrome C (#11940), ubiquitin (#43124), β-actin (#3700), ubiquitin (#3936), p-Survivin Thr34 (#8888), α-tubulin (#3873), p-Akt Ser473 (#4060), p-Wee1 Ser642 (#4910), His-tag (#12698), HA-tag (#2367), p-CDK1 Tyr15 (#4539), and p-CDK1 Thr161 (#9114) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against Ki67 (ab16667) and FbxL7 (#ab59149) were purchased from Abcam (Cambridge, UK). Flag-tag (F3165) antibody was obtained from Sigma Aldrich (St. Louis, MO). Lipofectamine® 2000 (Thermo Fisher Scientific) was used for plasmid transfection following the manufacturer’s instructions.